Compositions and methods for treating celiac sprue disease

ABSTRACT

Polypeptides, and methods for their use, are disclosed that have an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:1, are provided, wherein (a) the polypeptide degrades a PFQPQLPY (SEQ ID NO: 140) peptide and/or a PFPQPQQPF (SEQ ID NO: 68) at pH 4; (b) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and (c) the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424, 449, 461, 463, 105, 171, 172, 173, 174, and 456.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No. 15/575,159 filed Nov. 17, 2017, which is a national stage filing of PCT Application Serial No. PCT/US2016/036356 filed Jun. 8, 2016, which claims priority to U.S. Provisional Patent Application Ser. No. 62/172,557 filed Jun. 8, 2015, each incorporated by reference herein in its entirety.

BACKGROUND

Celiac sprue is a highly prevalent disease in which dietary proteins found in wheat, barley, and rye products known as ‘glutens’ evoke an immune response in the small intestine of genetically predisposed individuals. The resulting inflammation can lead to the degradation of the villi of the small intestine, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue and weight loss to abdominal distension, anemia, and neurological symptoms. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its' prevalence in the US and Europe is estimated at 0.5-1.0% of the population. In addition to celiac sprue, a significant fraction of the population is thought to suffer from the condition of non-celiac gluten sensitivity (NCGS), which is caused by the ingestion of gluten but is mechanistically distinct from celiac disease, though the symptoms are frequently indistinguishable from those of celiac sprue. The identification of suitable naturally-occurring enzymes as oral therapeutics for celiac disease and NCGS is difficult due to the stringent physical and chemical requirements to specifically and efficiently degrade gluten-derived peptides in the harsh and highly acidic environment of the human digestive tract. Since gluten peptides initiate the immune response immediately upon entering the intestines, it is imperative that any oral enzyme therapeutic for celiac disease break down these immunogenic gluten regions in the gastric compartment, thereby preventing these gluten peptides from causing intestinal damage due to inflammation.

SUMMARY OF THE INVENTION

In one aspect, the invention provides polypeptides comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or more identical to the amino acid sequence of SEQ ID NO: 1, wherein

(a) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 463, 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424, 449, 461, 105, 171, 172, 173, 174, and 456.

In one embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 463, 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424, 449, and 461. In another embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 221D/N/Q/H, 262E, 268S/T/A, 269L/T, 270A/T/V, 319A, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399Q, 402S/Q, 406S, 424K, 449E/N/Q, 461R, and 463A/L/M/Q/R/T/V. In a further embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 1 at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more residues selected from the group.

In one embodiment, the polypeptide comprises amino acid changes from SEQ ID NO: 1 at residues 399 and 449, such as amino acid changes 399Q and 449Q. In another embodiment, the polypeptide comprises amino acid changes 358S and 463T. In a further embodiment, the polypeptide comprises amino acid changes 262E, 269T, 354Q, 358S, 399Q, 449Q, and 463T. In another embodiment, the polypeptide comprises amino acid changes 319A, 368F, 399Q, 449Q, and 463T. In a further embodiment, the polypeptide comprises amino acid changes 262E, 269T, 270V, 354Q, 358S, 399Q, and 449Q. In a still further embodiment, the polypeptide comprises amino acid changes 262E, 269T, 320M, 354Q, 358S, 399Q, 449Q, and 463T. In another embodiment, the polypeptide comprises amino acid changes 319A, 320M, 368F, 399Q, 449Q, and 463T. In one embodiment, the polypeptides comprise an amino acid change from SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of 105, 171, 172, 173, 174, and 456, such as amino acid changes 105H; 171R A, or S; 172R, A, or S; 173R or S, 174S, and/or 456V.

In another aspect, the invention provides polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or more identical to the amino acid sequence of SEQ ID NO: 71, wherein

(a) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 71 at one or more residues selected from the group consisting of 274, 32, 73E, 79, 80, 81, 130A, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210, 213, 217, 235, 260, 267, and 272.

In one embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 71 at one or more residues selected from the group consisting of 274, 32, 73E, 79, 80, 81, 130A, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210, 213, 217, 235, 260, and 272.

In one embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 71 at one or more residues selected from the group consisting of 32D/N/Q/H, 73E, 79S/T/A, 80L/T, 81A/T/V, 130A, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210Q, 2135/Q, 217S, 235K, 260E/N/Q, 272R, and 274A/L/M/Q/R/TN. In a further embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 71 at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more residues selected from the group. In one embodiment, the polypeptide comprises amino acid changes from SEQ ID NO: 71 at residues 210 and 260, including but not limited to amino acid changes 210Q and 260Q. In another embodiment, the polypeptide comprises amino acid changes 169S and 274T. In a further embodiment, the polypeptide comprises amino acid changes 73E, 80T, 165Q, 169S, 210Q, 260Q, and 274T. In another embodiment, the polypeptide comprises amino acid changes 130A, 179F, 210Q, 260Q, and 274T. In a further embodiment, the polypeptide comprises amino acid changes 73E, 80T, 81V, 165Q, 169S, 210Q, and 260Q. In a still further embodiment, the polypeptide comprises amino acid changes 73E, 80T, 320M, 165Q, 169S, 210Q, 260Q, and 274T. In one embodiment, the polypeptide comprises amino acid changes 130A, 131M, 179F, 210Q, 260Q, and 274T. In another embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 71 at amino acid position 267, including but not limited to 267V.

In another embodiment, the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-42, 44-60 and 72-112, and 114-130 and 150-155. In a further embodiment, the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-42, 55-60 and 72-112, and 125-130 and 150-155.

In one embodiment, the polypeptides of the invention may further comprise a histidine tag at the C-terminus of the polypeptide. In another embodiment, the histidine tag comprises a cleavable histidine tag In a specific embodiment, the cleavable histidine tag may comprise or consist of the amino acid sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139). In another embodiment, the cleavable histidine tag may comprise the amino acid sequence X_(N)PQ(L/Q)PX_(N)HHHHHH (SEQ ID NO: 131), wherein X_(N) is an linker of between 1-25 amino acid residue. In a further embodiment, the cleavable histidine tag may comprise the amino acid sequence GSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 132).

In another aspect, the invention provides nucleic acids encoding the polypeptide of any embodiment of the invention. The invention further provides nucleic acid expression vector comprising the nucleic acids of the invention. The invention further provides recombinant host cells comprising the nucleic acid expression vectors of the invention. The invention also provides pharmaceutical composition, comprising the polypeptide, nucleic acid, nucleic acid expression vector, and/or the recombinant host cell of any embodiment of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides methods for treating celiac sprue or non-celiac gluten sensitivity (NCGS), comprising administering to an individual with celiac sprue or NCGS an amount effective to treat the celiac sprue or NCGS of a polypeptide or pharmaceutical composition of any embodiment of the invention. In one embodiment, the polypeptide or the pharmaceutical composition is administered orally.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Kuma030 is capable of rapidly and effectively degrading the immunogenic regions of gluten in gastric conditions. (A) The amount of gluten detected after a 60 minute incubation period with the indicated concentrations of either EPB2 and SCPEP (at a 1:1 ratio), Kuma010, or Kuma030 in gastric conditions, as measured by ELISA using the G12 antibody. The starting concentration of gluten was 10 mg mL-1 (10,000 ppm). Note that the Y-axis is plotted on a logarithmic scale. (B) The amount of gluten detected at 5 or 30 minutes after incubation with EPB2 and SCPEP at 400 μg mL-1, or Kuma030 at 10, 100, or 400 μg mL-1. The starting concentration of gluten was 10 mg mL-1. Samples were normalized to the amount of gluten measured after incubation with pepsin alone. ES: a 1:1 weight ratio of EPB2 and SCPEP. Asterisk indicates that the amount of gluten was below the level of quantification (5 ppm). (C) HPLC trace of full-length peptide (grey dotted lines) or breakdown products (black lines) of the immunodominant peptides from gliadin (W02-E07, W03-E07, 33mer), hordein (B08-E2E7), or secalin (R11-E4E7). The bottom of the figure shows the amino acid sequence of the 33mer peptide, position of known immunogenic epitopes (horizontal lines), location of the Kuma030 cleavage sites as determined by LCMS (vertical lines), and elution peaks of the resulting breakdown products (grey arrows). Immunodominant peptides displayed the following breakdown patterns: B08-E2E7: PQQPIPQ∥QPQPYPQ∥Q (SEQ ID NO: 61); R11-E4E5: QPFPQ∥QPEQIIPQ∥QP (SEQ ID NO:62); W02-E7: LQPFPQPQ∥LPYPQPQ (SEQ ID NO: 63); W03-E7: QPFPOPQ∥QPFPWQP (SEQ ID NO: 64). All peptide masses and elution times were confirmed by LCMS. Note that although the undigested W03-E07 peptide eluted at approximately the same time as the W03-E07 breakdown fragments, these are separate peaks, as determined by LCMS. aAU, arbitrary absorbance units.

FIG. 2. Gliadin treated with Kuma030 loses it immunostimulatory potential. (A-E) Purified gliadin was treated with Kuma030 at the specified concentration for 60 minutes at pH 4.0 at 37° C. in the presence of 0.6 mg mL-1 pepsin. After the gastric phase, the pH of the samples was increased, and samples were treated with chymotrypsin and TG2. Samples were then exposed to T cell lines from patients #1 (A), #2 (B), #3 (C), #4 (D), or #5 (E), in the presence of autologous irradiated B cell lines, and IFN-γ was measured by ELISA. Phytohemagglutenin (PHA) and a peptic-tryptic digest of gliadin (PT-Gliadin) were included as positive controls. Incubation of T cell lines with antigen-presenting cells in the absence of antigens acted as a negative control. (F) T cell stimulatory epitopes recognized by the T cells used in this assay and the predicted Kuma030 cleavage sites within these epitopes. Predicted Kuma030 cleavage sites are shown by a vertical Cleavage sites are predicted based on Kuma030 activity on gliadin peptides as presented in FIG. 1F (from top to bottom: SEQ ID NOs: 141, 142, 141, 142, 143, 144, 141, 142, 143, 144, 145, 145, 146, 145).

FIG. 3. Kuma030 is not toxic to T cells. Possible toxic effect of enzyme-treated gliadin was assessed in human peripheral blood mononuclear cells (PBMCs) from healthy donors that were stimulated with the mitogen phytohemagglutinin (PHA). (a,b) IFN-g production of T cells from healthy donor #1 (A) or healthy donor #2 (B). (C,D) T cell proliferation of T cells from healthy donor #1 (C) or healthy donor #2 (D). No effect on IFN-g production or cell proliferation was observed, except in the proliferation of healthy donor #2 (but not in the production of IFN-g). Since the reduction of PHA-stimulated cell proliferation was the same irrespective of the enzyme treatment, we conclude that this effect was not due to toxicity of the samples.

FIG. 4. Kuma030 efficiently degrades gluten in complex food matrices. (A) The amount of gluten remaining in whole wheat bread after a 30 minute incubation with either Kuma030 or a 1:1 combination of EPB2:SCPEP at the indicated enzyme concentrations. The starting concentration of gluten was 10,000 ppm. Note both axes are plotted on a logarithmic scale. (B) The amount of gluten remaining in a wheat beer after incubation with the indicated concentrations of either a 1:1 ratio of EPB2:SCPEP, or Kuma030, at 37° C. or 4° C., at 5, 15, or 60 minutes. Gluten was detected using the G12 ELISA method.

FIG. 5. Comparison of 26-mer and 33-mer degradative abilities of Kuma010, Kuma020, Kuma030, Kuma040, and Kuma050. Enzymes were incubated at 2 μg mL⁻¹ with 1 mg mL⁻¹ of either the 33mer peptide (FIG. 5a ) or the 26mer peptide (FIG. 5b ) for 60 minutes. Samples were taken at the indicated timepoints, and the concentrations of peptide breakdown products were measured. Breakdown products were: from the 33mer peptide (SEQ ID NO: 69), LPYPQPQF (SEQ ID NO: 137); from the 26mer peptide (SEQ ID NO: 70), QPYPQ (SEQ ID NO: 147). Activities are graphed as the ratio of the appropriate m/z signal to that of the internal standard.

FIG. 6. The G320M mutation improves activity by 2-4 fold. Shown is activity in degradation of immunogenic epitopes DQ2.5-glia-α1a (contains PQL) (FIG. 6a ), and DQ2.5-glia-ω1 (contains PQQ) (FIG. 6b ). Samples were taken at the indicated timepoints, and the concentration of peptide breakdown product PFPQPQ (SEQ ID NO: 148) was measured. Activities are graphed as the ratio of the appropriate m/z signal to that of the internal standard.

FIG. 7. P171R and H172R mutations do not appear to negatively affect activity. Enzymatic activities are shown for enzyme variants Kuma030, Kuma031, Kuma032, Kuma040, Kuma041, and Kuma042 on the immunogenic gliadin epitope DQ2.5-glia-α1a. Samples were taken at the indicated timepoints, and the concentration of peptide breakdown product PFPQPQ (SEQ ID NO: 148) was measured. Activities are graphed as the ratio of the appropriate m/z signal to that of the internal standard. All enzymes were purified by anion exchange.

FIG. 8. Kuma062 demonstrates a higher level of enzymatic activity than Kuma030 or Kuma040. Shown is activity on the immunogenic gliadin epitope DQ2.5-glia-ω1. FIG. 8a : detection of full-length DQ2.5-glia-ω1; FIG. 8b : detection of the PFPQPQ (SEQ ID NO: 148) breakdown product. Samples were taken at the indicated timepoints, and the concentration of either full-length peptide, or breakdown product PFPQPQ (SEQ ID NO: 148), was measured. Activities are graphed as the ratio of the appropriate m/z signal to that of the internal standard.

DETAILED DESCRIPTION

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides polypeptides comprising an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:1, wherein

(a) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424, 449, 461, 463, 105, 171, 172, 173, 174, and 456. In one embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 1 at one or more residues selected from the group consisting of 221, 262E, 268, 269, 270, 319A, 320, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399, 402, 406, 424, 449, 461, and 463.

SEQ ID NO: 1 (Kuma 011) Kuma011 (SEQ ID NO: 1) MSDMEKPWKE(10)GEEARAVLQG(20)HARAQAPQAV(30)DKGPVAGD ER(40)MAVTVVLRRQ(50)RAGELAAHVE(60)RQAAIAPHAR(70)EH LKREAFAA(80)SHGASLDDFA(90)ELRRFADAHG(100)LALDRANVA A(110)GTAVLSGPDD(120)AINRAFGVEL(130)RHFDHPDGSY (140)RSYLGEVTVP(150)ASIAPMIEAV(160)LGLDTRPVAR(170) PH(172)FRMQRRAE(180)GGFEARSQ(188)A A(190)APTAYTPLDV(200)AQAYQFPEGL(210)DGQGQCIAII (220)E(221/32)LGGGYDEAS(230/41)LAQYFASLGV(240/51) PAPQVVSVSV(250/61)DGASNQPTGD(260/71)PK(262/73)GPDG E(267/78)V(268/79)E(269/80)L(270/81)D(271/82)IEVAG ALAP(280/91)GAKFAVYFAP(290/101)DTTAGFLDAI(300/111) TTAIHDPTLK(310/121)PSVVSISWS(319/130)G(320/131)PED SWTSAAI(330/141)AAMNRAFLDA(340/151)AALGVTVLAA(350/ 161)AGDS(354/165)GSTG(358/169)GE(360/171)QDGLYHVH (368/179)FP(370/181)AASPYVLACG(380/191)GTRLVASGGR (390/201)IAQETVWND(399/210)G(400/211)PD(402/213)GG AT(406/217)GGGV(410/221)SRIFPLPAWQ(420/231)EHAN (424/235)VPPSAN(430/241)PGASSGRGVP(440/251)DLAGNAD PA(449/260)T(450/261)GYEVVIDGEA(460/271)T(461/272) VI(463/274)GGTS(467/278)AVAPLFAALVARINQKLGKAVGYLNP TLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRLLQA LLPSASQPQP

Kuma010 as reference herein is identical to Kuma011, but includes the histidine tag sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139) at its C-terminus.

Bold face residues represent the N-terminal portion present in the unprocessed polypeptide; non-bold faced font represents residues present in the processed version of the polypeptide. The numbers in parentheses indicate residue number; where there are two numbers separated by a “/”, the number on the left is the residue number in the unprocessed version, and the number on the right is the residue number in the processed version. SEQ ID NO:1 is the unprocessed version of Kuma011; SEQ ID NO:71 is the processed version of Kuma011.

As disclosed in the examples that follow, polypeptides according to this aspect of the invention are improved polypeptides for use, for example, in treating celiac sprue. The polypeptides are modified versions of either the processed version or the preprocessed version of the polypeptide of SEQ ID NO: 1 (KUMAMAX™, hereinafter referred to as Kuma010), which was disclosed as useful for treating celiac sprue (WO2013/023151). Polypeptides for treating celiac sprue are capable of degrading proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ believed responsible for the bulk of the immune response in most celiac sprue patients. The polypeptides of the present invention show superior activity in degrading peptides having a PQLP (SEQ ID NO: 65) or PQQP (SEQ ID NO: 66) motif (such as PFPQPQLPY (SEQ ID NO: 67) and/or PFPQPQQPF (SEQ ID NO: 68)), which are substrates representative of gliadin) at pH 4 compared to Kuma011 and other polypeptides disclosed as useful for treating celiac sprue (WO2015/023728), and/or are shown to improve production of the polypeptides. Thus, the polypeptides of the invention constitute significantly improved therapeutics for treating celiac sprue.

Thus, the polypeptides of the invention degrade a PFPQPQLPY (SEQ ID NO: 67) peptide and/or a PFPQPQQPF (SEQ ID NO: 68) peptide at pH 4, as well as LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69) and/or FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70).

Polypeptides of the first aspect of the invention comprise preprocessed versions of the polypeptide enzymes of the invention.

In a second aspect, the invention provides polypeptides comprising an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:71, wherein

(a) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; and

(b) the polypeptide comprises an amino acid change from SEQ ID NO: 71 at one or more residues selected from the group consisting of 32, 73E, 79, 80, 81, 130A, 131, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210, 213, 217, 235, 260, 267, 272, and 274. In one embodiment, the polypeptide comprises an amino acid change from SEQ ID NO: 71 at one or more residues selected from the group consisting of 32, 73E, 79, 80, 81, 130A, 131, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210, 213, 217, 235, 260, 272, and 274.

Polypeptides of the first aspect of the invention comprise processed versions of the polypeptide enzymes of the invention, and also degrade a PFPQPQLPY (SEQ ID NO: 67) peptide and/or a PFPQPQQPF (SEQ ID NO: 68) peptide at pH 4, as well as LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69) and/or

FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70).

As used herein, “at least 75% identical” means that the polypeptide differs in its full length amino acid sequence by 25% or less (including any amino acid substitutions, deletions, additions, or insertions) from the polypeptide defined by SEQ ID NO: 1 or SEQ ID NO: 71.

In various embodiments of any aspect of the polypeptides of the invention, the polypeptides comprise or consist of an amino acid sequence at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence according to SEQ ID NO: 1 (preprocessed) or SEQ ID NO:71 (processed).

The polypeptide of any aspect of the polypeptides of the invention may comprises an amino acid change from SEQ ID NO: 1 or SEQ ID NO:71 at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all 24 (depending on the embodiment) of the recited residues.

In one embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises one or more amino acid changes from SEQ ID NO: 1 at one or more residues selected from the group consisting of 221D/N/Q/H, 262E, 2685/T/A, 269L/T, 270A/T/V, 319A, 354E/Q/R/Y, 358S/Q/T, 368F/Q, 399Q, 402S/Q, 406S, 424K, 449E/N/Q, 461R, and 463A/L/M/Q/R/T/V. As used throughout, the number indicates the residue number in the SEQ ID NO:1 or SEQ ID NO:71 polypeptide sequence, and the single letter amino acid abbreviations to the right of the number indicate the possible amino acid substitutions compared to the amino acid residue present at that position in SEQ ID NO:1 or 71.

In another embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises amino acid changes from SEQ ID NO: 1 at residues 399 and 449. In one embodiment, the polypeptide comprises amino acid changes 399Q and 449Q.

In a further embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises 358S and 463T. These polypeptide are extensively characterized in the examples that follow, as exemplified by the polypeptide designated as Kuma020 and variants thereof.

In one embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises 262E, 269T, 354Q, 358S, 399Q, 449Q, and 463T. These polypeptide are extensively characterized in the examples that follow, as exemplified by the polypeptide designated as Kuma030 and variants thereof. In another embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises 319A, 368F, 399Q, 449Q, and I463T. These polypeptide are extensively characterized in the examples that follow, as exemplified by the polypeptide designated as Kuma040 and variants thereof. In a further embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises 262E, 269T, 270V, 354Q, 358S, 399Q, and A449Q. These polypeptide are extensively characterized in the examples that follow, as exemplified by the polypeptide designated as Kuma050 and variants thereof. In one embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises 262E, 269T, 320M, 354Q, 358S, 399Q, 449Q, and 463T. These polypeptide are extensively characterized in the examples that follow, as exemplified by the polypeptide designated as Kuma060 and variants thereof. In a still further embodiment of the polypeptides of the first aspect of the invention, the polypeptide comprises, 319A, 320M, 368F, 399Q, 449Q, and 463T. These polypeptide are extensively characterized in the examples that follow, as exemplified by the polypeptide designated as Kuma070 and variants thereof.

In another embodiment of the polypeptides of the first aspect of the invention, the polypeptides comprise an amino acid change from SEQ ID NO: 1 at one or more amino acid positions selected from the group consisting of 105, 171, 172, 173, 174, and 456. In one embodiment, the amino acid change is 105H; 171R A, or S; 172R, A, or S; 173R or S, 174S, and/or 456V. In another embodiment, the amino acid change is 171R, 172R, and/or 456V.

In one embodiment of the polypeptides of the second aspect of the invention the polypeptide comprises one or more amino acid change from SEQ ID NO: 71 at one or more residues selected from the group consisting of 32D/N/Q/H, 73E, 795/T/A, 80L/T, 81A/TN, 130A, 165E/Q/R/Y, 169S/Q/T, 179F/Q, 210Q, 213S/Q, 217S, 235K, 260E/N/Q, 272R, and 274A/L/M/Q/R/T/V. In another embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises amino acid changes from SEQ ID NO: 71 at residues 210 and 260. In a further embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises amino acid changes 210Q and 260Q. In one embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises 169S and 274T. (Kuma 20 genus) In another embodiment of the polypeptides of the second aspect of the invention the polypeptide comprises 73E, 80T, 165Q, 169S, 210Q, 260Q, and 274T. (Kuma 30 genus) In a further embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises 130A, 179F, 210Q, 260Q, and 274T. (Kuma 40 genus) In a still further embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises 73E, 80T, 81V, 165Q, 169S, 210Q, and 260Q. (Kuma 50 genus) In one embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises 73E, 80T, 320M, 165Q, 169S, 210Q, 260Q, and 274T. (Kuma 60 genus) In another embodiment of the polypeptides of the second aspect of the invention, the polypeptide comprises 130A, 131M, 179F, 210Q, 260Q, and 274T. (Kuma 70 genus) In a still further embodiment of the polypeptides of the second aspect of the invention, the polypeptides comprise an amino acid change from SEQ ID NO: 71 at one or more amino acid positions selected from the group consisting of 267. In one embodiment, the amino acid change is, 267V.

In a further embodiment of the polypeptides of any aspect of the invention, the polypeptides further comprise a histidine tag at the C-terminus of the polypeptide, to facilitate isolation of the polypeptide. Any suitable histidine tag can be used; in one embodiment the tag is linked to a TEV protease cut sit (ENLYFQS) (SEQ ID NO: 149) to allow for its efficient removal with TEV protease after purification, for example, the tag may comprise or consist of the amino acid sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139). In another embodiment, the histidine tag is a. cleavable histidine tag, permitting easier removal of the His-tag. In one embodiment, the cleavable histidine tag comprises the amino acid sequence X_(N)PQ(L/Q)PX_(N)HHHHHH (SEQ ID NO: 131), wherein X_(N) is an linker of between 1-25 amino acid residues. In one non-limiting example, the cleavable histidine tag comprises the amino acid sequence GSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 132).

In one embodiment of any aspect of the polypeptides of the invention, amino acid substitutions compared to SEQ ID NO: 1 or SEQ ID NO: 71 may comprise one or more of the substitutions noted in Tables 1 or 2. Substitutions at these positions were found to be generally well-tolerated (i.e. generally result in minor to no effects on activity), and in some cases to increase the activity of the polypeptides of the invention by no more than 20%.

TABLE 1 Possible Amino Acid Substitutions at Position Relative to Kuma10. Residue number (preprocessed/processed) Residue 221/32 D, N, Q, H 261/72 A, R, N, D, C, Q, E, G, H, I, L, K, M, S, T, W, Y, V 262/73 A, R, N, D, C, Q, E, G, H, I, L, M, F, T, W, Y, V 264/75 A, N, D, C, Q, E, G, S, T, Y 266/77 A, C, S 268/79 S, T 269/80 L, T 270/81 A, R, N, D, C, Q, E, G, I, K, S, T, V 317/128 A, N, C, G, T, V 318/129 A, R, N, D, C, Q, E, G, H, L, K, M, F, S, T, Y, V 319/130 A, N, D, C, Q, H, M, T 320/131 A, R, N, D, C, Q, K, M, S 350/161 N, D, C, G, S, T 351/162 G, S 353/164 A, R, N, C, Q, E, G, I, K, M, S, T, V 354/165 A, R, N, D, C, Q, E, G, H, L, K, M, F, T, W, Y 358/169 A, S, N, Q, T 368/179 A, R, N, C, Q, E, G, K, M, F, S, T, W, Y 397/208 A, C, F, Y 399/210 Q, N 402/213 Q, N, S 406/217 S 424/235 K 446/257 G, S 448/259 A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y, V 449/260 Q, E, G, N 456/267 A, N, D, C, Q, E, G, H, L, S, T, V 461/272 R 463/274 A, R, N, D, C, Q, E, G, H, L, K, M, F, S, T, W, Y, V 464/277 A, N, D, C, S, 466/279 D, C, G, S

In another embodiment of any aspect of the polypeptides of the invention, amino acid substitutions compared to SEQ ID NO: 1 or SEQ ID NO: 71 may comprise one or more of the substitutions noted in Table 2.

Residue number (preprocessed/processed) Residue 221/32 D, N, Q, H 261/72 S 262/73 A, R, N, D, Q, E, G, L, M, T 264/75 A 268/79 S, T 269/80 L, T 270/81 A, T, V 317/128 A, T 319/130 A 354/165 A, R, N, D, Q, E, K, T, Y 358/169 A, S, N, Q, T 368/179 A, N, D, Q, E, S, T 402/213 Q, S 406/217 S 424/235 K 446/257 S 449/260 Q, N, A 456/267 V 461/272 R 463/274 A, R, Q, L, M, T, V

In another embodiment of any aspect of the polypeptides of the invention, amino acid at each residue of the polypeptides of the invention may be as noted in Table 3, which lists all of the possible mutations at each position in the polypeptide enzymes as predicted by computational mutagenesis analysis. As described in the examples that follow, mutations were tested at each position found in the active site (residues 261-264, 266-267, 270, 317-320, 350-354, 368, 397, 403-404, 446, 448, 456, and 463-468) using degenerate primers to test the effects of various amino acid substitutions on activity; those that did not interfere with activity can be incorporated in the polypeptides of the invention, as reflected in Table 3.

TABLE 3 Possible Amino Acids at Residues Relative to Kuma 010 Full Length Mature Amino Acid Possibilities 190 1 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 191 2 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, PRO, SER, THR, TRP, VAL 192 3 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, PRO, SER, TRP, TYR 193 4 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 194 5 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 195 6 ALA, ASN, CYS, GLN, HIS, LEU, MET, PHE, THR, TYR 196 7 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, THR, TRP, TYR 197 8 ALA, GLY, PRO, SER 198 9 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 199 10 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 200 11 ALA, ASN, ASP, CYS, GLY, ILE, SER, THR, VAL 201 12 ALA, CYS, GLY, SER 202 13 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 203 14 ALA, GLY, SER 204 15 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR 205 16 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 206 17 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 207 18 ALA, CYS, GLN, GLU, GLY, LYS, PRO, SER, THR, TRP 208 19 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 209 20 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 210 21 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER, THR, VAL 211 22 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, THR, TYR 212 23 GLY 213 24 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 214 25 GLY 215 26 ALA, ASN, ASP, CYS, GLN, GLU, GLY, SER, THR 216 27 ALA, ASN, ASP, CYS, GLN, GLY, SER, THR, VAL 217 28 ALA, CYS, ILE, LEU, SER, THR, VAL 218 29 ALA, GLY, SER 219 30 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, MET, SER, THR, VAL 220 31 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR, VAL 221 32 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR, VAL 222 33 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, SER, THR, VAL 223 34 ALA, ARG, ASN, ASP, CYS, GLU, GLY, LYS, MET, SER 224 35 GLY 225 36 GLY 226 37 ALA, ARG, ASN, ASP, CYS, GLU, GLY, HIS, LEU, PHE, SER, THR, TRP, TYR 227 38 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LYS, MET, SER 228 39 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 229 40 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 230 41 ALA, GLY, SER 231 42 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, SER, THR 232 43 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 233 44 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 234 45 ALA, ASN, CYS, GLY, HIS, PHE, SER, TYR 235 46 ALA, ASN, ASP, CYS, HIS, MET, PHE, SER, THR, TRP, TYR 236 47 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 237 48 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 238 49 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, MET, SER, THR, VAL 239 50 GLY 240 51 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, SER, THR, TYR, VAL 241 52 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 242 53 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PRO, SER, THR, VAL 243 54 ALA, GLY, PRO, SER 244 55 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 245 56 ALA, ASN, CYS, GLY, SER, THR, VAL 246 57 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 247 58 ALA, ARG, ASP, CYS, GLY, ILE, LYS, MET, PRO, SER 248 59 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 249 60 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, PRO, SER, THR 250 61 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR, VAL 251 62 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 252 63 ASN, ASP, GLY, SER 253 64 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, THR, TRP 254 65 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 255 66 ALA, ARG, ASN, ASP, CYS, MET, SER, THR 256 67 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 257 68 ALA, ARG, ASN, CYS, GLN, GLU, GLY, ILE, LYS, MET, PRO, SER, THR, VAL 258 69 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 259 70 GLY 260 71 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 261 72 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, TRP, TYR, VAL 262 73 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 263 74 GLY 264 75 ALA, ASN, ASP, CYS, GLN, GLU, GLY, PRO, SER, THR, TRP 265 76 ALA, ASN, ASP, CYS, GLN, GLU, GLY, SER, THR, VAL 266 77 ALA, CYS, GLY, SER 267 78 GLU 268 79 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 269 80 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 270 81 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, SER, THR, VAL 271 82 ASP 272 83 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, MET, SER, THR, VAL 273 84 ALA, ASN, ASP, CYS, GLN, GLU, GLY, SER, THR 274 85 ALA, ASN, ASP, CYS, GLY, ILE, SER, THR, VAL 275 86 ALA, CYS, GLY, SER 276 87 GLY 277 88 ALA, GLY, SER 278 89 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, MET, SER, THR, VAL 279 90 ALA, GLY, SER 280 91 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, MET, PHE, PRO, SER, TRP, TYR 281 92 GLY 282 93 ALA, GLY, SER 283 94 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 284 95 CYS, HIS, ILE, LEU, MET, PHE, THR, TYR, VAL 285 96 ALA, GLY, SER 286 97 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 287 98 ALA, ASN, ASP, CYS, GLN, HIS, LEU, PHE, SER, TYR 288 99 HIS, PHE 289 100 ALA, GLY, SER 290 101 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 291 102 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL, 292 103 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 293 104 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, VAL 294 105 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 295 106 GLY 296 107 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, VAL 297 108 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 298 109 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 299 110 ALA, GLY, SER 300 111 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 301 112 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, PHE, SER, THR, VAL 302 113 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, SER, THR, TRP, VAL 303 114 ALA, GLY, SER 304 115 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, SER, THR, VAL 305 116 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 306 117 ALA, ASN, ASP, SER 307 118 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 308 119 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 309 120 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 310 121 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 311 122 ALA, CYS, GLY, PRO, SER 312 123 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, THR, TRP, TYR 313 124 ALA, CYS, GLY, ILE, SER, THR, VAL 314 125 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, SER, THR, VAL 315 126 ALA, CYS, GLY, SER, THR 316 127 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, MET, SER, THR, VAL 317 128 ALA, ASN, CYS, GLY, SER, THR, VAL 318 129 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 319 130 ALA, ASN, ASP, CYS, GLN, GLY, HIS, MET, SER, THR 320 131 ALA, ARG, ASN, ASP, CYS, GLN, GLY, LYS, MET, SER 321 132 ALA, CYS, GLY, PRO, SER 322 133 ALA, ASP, CYS, GLN, GLU, GLY, LEU, SER 323 134 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, VAL, 324 135 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 325 136 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, TRP, TYR 326 137 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 327 138 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 328 139 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 329 140 ALA, ASP, CYS, GLY, SER 330 141 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 331 142 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 332 143 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 333 144 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, VAL 334 145 ALA, ARG, ASN, ASP, CYS, GLU, GLY, MET, SER, THR, VAL 335 146 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 336 147 ALA, ARG, CYS, GLN, GLU, GLY, MET, SER 337 148 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 338 149 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 339 150 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 340 151 ALA, ASN, ASP, GLY, SER 341 152 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, SER, THR, VAL 342 153 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 343 154 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 344 155 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 345 156 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 346 157 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, THR 347 158 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, PRO, SER, THR, VAL 348 159 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, SER, THR, VAL 349 160 ALA, CYS, GLY, SER, THR 350 161 ALA, ASN, ASP, CYS, GLY, SER, THR 351 162 ALA, GLY, SER 352 163 GLY 353 164 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, SER, THR, VAL 354 165 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 355 166 GLY 356 167 ALA, GLY, SER 357 168 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, MET, SER, THR, VAL 358 169 ALA, GLY, SER 359 170 ASN, GLY 360 171 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 361 172 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 362 173 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 363 174 ASN, ASP, GLY, SER 364 175 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 365 176 ALA, ARG, ASN, ASP, CYS, GLY, HIS, MET, PHE, SER, THR, TRP, TYR 366 177 ALA, ASN, ASP, CYS, HIS, LYS, SER 367 178 ALA, ASP, CYS, GLY, SER, THR, VAL 368 179 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, THR, TRP, TYR 369 180 ALA, CYS, HIS, PHE, SER, TYR 370 181 ALA, ASP, CYS, GLY, PRO, SER 371 182 ALA, GLY, SER 372 183 ALA, CYS, GLY, SER 373 184 ALA, GLY, SER 374 185 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, TRP, VAL 375 186 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 376 187 ALA, ASN, ASP, CYS, GLY, HIS, ILE, LEU, SER, THR, VAL 377 188 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, VAL 378 189 ALA, GLY, SER 379 190 ALA, ASP, CYS, GLY, SER, THR 380 191 GLY 381 192 GLY 382 193 ALA, CYS, GLY, SER, THR 383 194 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 384 195 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, SER, THR 385 196 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, TRP, VAL 386 197 ALA, CYS, GLY, MET, SER, THR 387 198 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 388 199 ASN, ASP, GLY, LYS, SER 389 200 GLY 390 201 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 391 202 ALA, ASN, ASP, CYS, GLN, GLY, ILE, MET, PRO, SER, THR, VAL 392 203 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 393 204 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 394 205 ALA, CYS, GLN, GLU, GLY, SER, THR 395 206 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, SER, THR, VAL 396 207 ALA, CYS, GLY, SER, THR, VAL 397 208 ALA, CYS, PHE, TRP, TYR 398 209 ARG, ASN, ASP, CYS, GLN, MET, SER 399 210 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, LYS, MET, SER 400 211 GLY 401 212 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 402 213 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 403 214 GLY 404 215 GLY 405 216 ALA, GLY, SER 406 217 ALA, CYS, GLY, SER, THR 407 218 GLY 408 219 GLY 409 220 GLY 410 221 ALA, ASN, CYS, GLY, ILE, SER, THR, VAL 411 222 ALA, GLY, SER 412 223 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, VAL 413 224 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, PHE, SER, THR, TYR, VAL 414 225 ALA, ASN, CYS, GLN, GLU, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 415 226 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 416 227 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, PRO, SER, THR, VAL 417 228 ALA, CYS, GLN, GLU, GLY, MET, PRO, SER, THR 418 229 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 419 230 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, PHE, SER, TRP, TYR 420 231 GLN, GLU 421 232 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 422 233 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 423 234 ALA, GLY, SER 424 235 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 425 236 ALA, CYS, GLY, PRO, SER, THR, VAL 426 237 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 427 238 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 428 239 ALA, ASN, ASP, CYS, GLN, GLU, GLY, SER, THR, VAL 429 240 ALA, ASN, ASP, CYS, GLY, SER 430 241 ALA, ASN, ASP, CYS, GLY, SER, THR 431 242 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 432 243 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 433 244 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 434 245 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 435 246 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 436 247 GLY 437 248 ALA, ARG, ASN, CYS, GLN, SER, THR 438 249 GLY 439 250 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, MET, SER, THR, VAL 440 251 ALA, GLY, PRO, SER 441 252 ASP 442 253 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER, THR 443 254 ALA, GLY, SER 444 255 ALA, GLY 445 256 ALA, ASN, ASP, CYS, GLY, SER 446 257 ALA, GLY, SER 447 258 ALA, ASN, ASP, CYS, GLY, SER, THR 448 259 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 449 260 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 450 261 ALA, ASN, ASP, CYS, GLY, HIS, SER, THR 451 262 GLY 452 263 ALA, ASN, CYS, GLN, HIS, ILE, LEU, PHE, SER, THR, TYR, VAL 453 264 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 454 265 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 455 266 ALA, ARG, ASN, ASP, CYS, GLU, GLY, HIS, ILE, MET, PHE, SER, THR, TRP, TYR, VAL 456 267 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, SER, THR, VAL 457 268 ALA, ASN, ASP, CYS, GLY, ILE, MET, SER, THR, TRP, VAL 458 269 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LYS, MET, SER 459 270 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 460 271 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TYR, VAL, 461 272 ALA, ASN, ASP, CYS, GLN, GLY, HIS, LYS, MET, SER, THR 462 273 ALA, ARG, ASN, ASP, CYS, GLN, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 463 274 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL, 464 275 GLY 465 276 GLY 466 277 ALA, ASN, ASP, CYS, GLY, SER, THR 467 278 SER 468 279 ALA, ASP, CYS, GLY, SER 469 280 ALA, ASN, ASP, CYS, GLY, SER, THR, VAL 470 281 ALA, GLY, SER 471 282 ALA, CYS, GLY, PRO, SER 472 283 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, MET, SER, THR, VAL 473 284 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 474 285 ALA, GLY, SER 475 286 ALA, GLY, SER 476 287 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, SER, THR, VAL 477 288 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, SER, THR, VAL 478 289 ALA, GLY, SER 479 290 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, SER, THR, TRP, TYR 480 291 ALA, ARG, ASN, ASP, CYS, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 481 292 ALA, ASN, ASP, CYS, GLN, GLU, GLY, MET, SER 482 293 ALA, GLN, GLU, HIS, LYS, THR 483 294 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, PHE, SER, TRP, TYR 484 295 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 485 296 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, TRP, TYR, VAL 486 297 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 487 298 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PRO, SER, THR, TRP, VAL 488 299 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, SER, THR, VAL 489 300 GLY 490 301 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 491 302 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, VAL 492 303 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, VAL 493 304 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 494 305 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 495 306 ALA, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, MET, SER, THR 496 307 ALA, HIS, PHE, SER, THR, TYR 497 308 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 498 309 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER, THR 499 310 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR 500 311 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 501 312 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 502 313 ALA, ASN, ASP, CYS, GLY, ILE, MET, SER, THR, VAL 503 314 ALA, ASN, ASP, CYS, HIS, LEU, MET, PHE, SER, THR, TYR, VAL 504 315 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 505 316 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PRO, SER, THR, TRP, VAL 506 317 ALA, ASN, ASP, CYS, GLN, GLY, ILE, SER, THR, VAL 507 318 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 508 319 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 509 320 GLY 510 321 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, SER, THR, TRP, TYR 511 322 ALA, ASN, ASP, CYS, GLY, SER 512 323 ALA, ASN, ASP, CYS 513 324 ALA, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LYS, MET, SER, THR, VAL 514 325 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 515 326 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LYS, MET, SER 516 327 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 517 328 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 518 329 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 519 330 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 520 331 HIS, PHE, THR, TRP, TYR 521 332 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 522 333 ALA, GLY, SER 523 334 CYS, GLY, HIS, LYS, MET, PHE, SER, TYR 524 335 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, PRO, SER, THR, TRP, TYR, VAL 525 336 GLY 526 337 HIS, PHE, TRP 527 338 ALA, ASN, ASP, CYS, SER 528 339 ALA, GLY, PRO, SER 529 340 ALA, ASP, CYS, GLY, SER, THR 530 341 ALA, ASN, CYS, GLY, SER, THR, VAL 531 342 GLY 532 343 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, LYS, MET, SER 533 344 GLY 534 345 ALA, CYS, GLY, SER, THR 535 346 ALA, CYS, GLY, PRO, SER, THR 536 347 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, ILE, LEU, LYS, MET, PHE, SER, THR, TYR, VAL 537 348 GLY 538 349 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 539 350 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, LEU, LYS, MET, PHE, SER, THR, TRP, TYR 540 351 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, LYS, SER, THR, VAL 541 352 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, LEU, LYS, MET, SER, THR 542 353 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP, TYR, VAL 543 354 ALA, ARG, CYS, GLN, GLU, GLY, MET, SER, THR 544 355 ALA, ASN, ASP, CYS, GLN, GLU, GLY, LEU, MET, SER, THR 545 356 ALA, ARG, ASN, ASP, CYS, GLN, GLU, GLY, HIS, ILE, LEU, LYS, MET, PHE, SER, THR, TRP 546 357 Any residue 547 358 Any residue 548 359 Any residue 549 360 Any residue 550 361 Any residue 551 362 Any residue 552 363 Any residue 553 364 Any residue

In a further embodiment, the polypeptides of the invention comprise or consist of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-42, 44-60 and 72-112, and 114-130 and 150-155, shown below. These polypeptides have increased activity relative to Kuma010, as shown in the examples that follow, or provide for improved production of the polypeptides. In one embodiment, the polypeptides comprise or consist of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2-42, 55-60 and 72-112, and 125-130 and 150-155; these polypeptides all show improved activity to Kuma010.

The N-terminal domain is in bold font, and changes relative to Kuma 011 are noted next to the polypeptide name. In all cases, the polypeptides described below may further comprise a histidine tag at the C-terminus. Any suitable histidine tag can be used; Any suitable histidine tag can be used; in one embodiment the tag is linked to a TEV protease cut sit (ENLYFQS) (SEQ ID NO: 149) to allow for its efficient removal with TEV protease after purification, for example, the tag may comprise or consist of the amino acid sequence GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139). In another embodiment, a cleavable histidine tag may be incorporated at the C-terminus, comprising the amino acid sequence X_(N)PQ(L/Q)PX_(N)HHHHHH (SEQ ID NO: 131), wherein X_(N) is an linker of between 1-25 amino acid residues. In one non-limiting example, the cleavable histidine tag may comprise the amino acid sequence GSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 132).

Kuma011-K262E MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAI HDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 2 (unprocessed), SEQ ID NO: 72 (processed)) Kuma011-V268A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEAELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 3 (unprocessed), SEQ ID NO: 73 (processed)) Kuma011-V268S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGESELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAI HDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 4 (unprocessed), SEQ ID NO: 74 (processed)) Kuma011-V268T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGETELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAI HDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 5 (unprocessed), SEQ ID NO: 75 (processed)) Kuma011-E269L MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVLLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 6 (unprocessed), SEQ ID NO: 76 (processed)) Kuma011-E269T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 7 (unprocessed), SEQ ID NO: 77 (processed)) Kuma011-L270A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVEADIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 8 (unprocessed), SEQ ID NO: 78 (processed)) Kuma011-L270T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVETDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 9 (unprocessed), SEQ ID NO: 79 (processed)) Kuma011-L270V MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVEVDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 10 (unprocessed), SEQ ID NO: 80 (processed)) Kuma011-G319A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWAGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 11 (unprocessed), SEQ ID NO: 81 (processed)) Kuma011-S354A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDAGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 12 (unprocessed), SEQ ID NO: 82 (processed)) Kuma011-S354E MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDEGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 13 (unprocessed), SEQ ID NO: 83 (processed)) Kuma011-S354O MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 14 (unprocessed), SEQ ID NO: 84 (processed)) Kuma011-S354R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDRGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 15 (unprocessed), SEQ ID NO: 85 (processed)) Kuma011-S354Y MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDYGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 16 (unprocessed), SEQ ID NO: 86 (processed)) Kuma011-G358N MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTNGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 17 (unprocessed), SEQ ID NO: 87 (processed)) Kuma011-G358S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTSGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 18 (unprocessed), SEQ ID NO: 88 (processed)) Kuma011-G358Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTQGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 19 (unprocessed), SEQ ID NO: 89 (processed)) Kuma011-G358T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTTGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 20 (unprocessed), SEQ ID NO: 90 (processed)) Kuma011-H368F MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVFFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 21 (unprocessed), SEQ ID NO: 91 (processed)) Kuma011-H368Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVQFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 22 (unprocessed), SEQ ID NO: 92 (processed)) Kuma011-D399Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 23 (unprocessed), SEQ ID NO: 93 (processed)) Kuma011-D402S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPSGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 24 (unprocessed), SEQ ID NO: 94 (processed)) Kuma011-D402Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPQGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 25 (unprocessed), SEQ ID NO: 95 (processed)) Kuma011-T406S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPQGGASGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 26 (unprocessed), SEQ ID NO: 96 (processed)) Kuma011-N424K MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNQGPQGGASGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 27 (unprocessed), SEQ ID NO: 97 (processed)) Kuma011-A449E MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPETGYEVVIDGEATVIGGTSAVAPLF AALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPC TGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 28 (unprocessed), SEQ ID NO: 98 (processed)) Kuma011-A449Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPQTGYEVVIDGEATVIGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 29 (unprocessed), SEQ ID NO: 99 (processed)) Kuma011-I456V MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVVDGEATVTGGTSAVAP LFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWD PCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 30 (unprocessed), SEQ ID NO: 100 (processed)) Kuma011-T461R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEARVTGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 31 (unprocessed), SEQ ID NO: 101 (processed)) Kuma011-I463A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVAGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 32 (unprocessed), SEQ ID NO: 102 (processed)) Kuma011-I463L MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVLGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 33 (unprocessed), SEQ ID NO: 103 (processed)) Kuma011-I463M MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVMGGTSAVAP LFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWD PCTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 34 (unprocessed), SEQ ID NO: 104 (processed)) Kuma011-I463Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVQGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 35 (unprocessed), SEQ ID NO: 105 (processed)) Kuma011-I463R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVRGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 36 (unprocessed), SEQ ID NO: 106 (processed)) Kuma011-I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVTGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 37 (unprocessed), SEQ ID NO: 107 (processed)) Kuma011-I463V MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTGGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVVGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 38 (unprocessed), SEQ ID NO: 108 (processed)) Kuma023: G368S; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQR AGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALD RANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEA VLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLGVPAPQVV SVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTA IHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLDAAALGVTVLAAAGDSGSTSGE QDGLYHVHFPAASPYVLACGGTRLVASGGRIAQETVWNDGPDGGATGGGVSRIFPL PAWQEHANVPPSANPGASSGRGVPDLAGNADPATGYEVVIDGEATVTGGTSAVAPL FAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDP CTGLGSPIGVRLLQALLPSASQPQP (SEQ ID NO: 39 (unprocessed), SEQ ID NO: 109 (processed))

Kuma 020 as referenced in the examples is the Kuma023 polypeptide that includes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139).

Kuma021: G368S; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 154 (unprocessed), SEQ ID NO: 155 (processed)) Kuma031: K262E; E269T; S354Q; G358S; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 40 (unprocessed), SEQ ID NO: 110 (processed))

Kuma 030 as referenced in the examples is the Kuma031 polypeptide that includes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139).

Kuma041: S319A; H368F; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWAGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 41 (unprocessed), SEQ ID NO: 111 (processed))

Kuma 040 as referenced in the examples is the Kuma041 polypeptide that includes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139).

Kuma051: K262E; E269T; L270V; S354Q; G358S; D399Q; A449Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVTVDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 42 (unprocessed), SEQ ID NO: 112 (processed))

Kuma 050 as referenced in the examples is the Kuma051 polypeptide that includes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139).

Kuma022: P171R; H172R; G368S; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 55 (unprocessed), SEQ ID NO: 125 (processed)) Kuma032: P171R; H172R; K262E; E269T; S354Q; G358S; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 56 (unprocessed), SEQ ID NO: 126 (processed)) Kuma042: P171R; H172R; S319A; H368F; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWAGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 57 (unprocessed), SEQ ID NO: 127 (processed)) Kuma052: P171R; H172R; K262E; E269T; L270V; S354Q; G358S; D399Q; A449Q MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVTVDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 58 (unprocessed), SEQ ID NO: 128 (processed)) Kuma061: K262E; E269T; S354Q; G358S; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYF APDTTAGFLDAITTAIHDPTLKPSVVSISWSMPEDSWTSAAIAAMNRAF LDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVA SGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASS GRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKL GKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLG SPIGVRLLQALLPSASQPQP (SEQ ID NO: 150 (unprocessed), SEQ ID NO: 151 (processed))

Kuma 060 as referenced in the examples is the Kuma061 polypeptide that includes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139).

Kuma062: P171R; H172R; K262E; E269T; S354Q; G358S; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSMPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 59 (unprocessed), SEQ ID NO: 129 (processed)) Kuma071: S319A; H368F; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWAMPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 152 (unprocessed), SEQ ID NO: 153 (processed))

Kuma 070 as referenced in the examples is the Kuma071 polypeptide that includes the following C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139).

Kuma072: P171R; H172R; S319A; H368F; D399Q; A449Q; I463T MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVTLDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWAMPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVFFPAASPYVLACGGTRLVASGG RIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 60 (unprocessed), SEQ ID NO: 130 (processed))

As described in Table 5, the changes made to Kuma010/011 have significant effect on the catalytic activity of the design proteins. Table 5 lists the effectiveness of individual mutations in catalyzing the degradation of various gliadin peptide sequences. The examples provide further data regarding specific individual and combination mutants.

TABLE 5 % Improvement on % Improvement Position A.A. relative PFPQPQLPY on (Full Position Kuma010 to (SEQ ID NO: PFPQPQQPF (SEQ Length) (Truncated) A.A. Kuma010/011 67) ID NO: 68) 221 32 E D, N, Q, H 105% ND 262 73 K E 109% 110% 268 79 V A 107% 89% 268 79 V S 104% 83% 268 79 V T 127% 105% 269 80 E L 113% 84% 269 80 E T 263% 191% 270 81 L A 203% 92% 270 81 L T 307% 29% 270 81 L V 474% 61% 319 130 S A 154% 184% 354 165 S A 152% 140% 354 165 S E 124% 120% 354 165 S Q 145% 141% 354 165 S R 109% 82% 354 165 S Y 46% 105% 358 169 G N 120% 99% 358 169 G S 331% 224% 358 169 G Q 147% 149% 358 169 G T 283% 128% 368 179 H F 334% 104% 368 179 H Q 199% 195% 399 210 D Q 149% 208% 402 213 D S 94% 108% 402 213 D Q 164% 111% 406 217 T S 84% 101% 424 235 N K 285% ND 449 260 A E 149% 208% 449 260 A N 119% 118% 461 272 T R 120% 86% 463 274 I A 51% 234% 463 274 I L 124% 22% 463 274 I M 123% 53% 463 274 I Q 129% 69% 463 274 I R 29% 110% 463 274 I T 130% 239% 463 274 I V 256% 141% Improved Production Mutations

Mutations that improve production may provide improvements in one of three categories: 1. Altering purification method; 2. increase in yield; and 3. decreasing the probability that enzymatic self-processing would occur during purification, thereby simplifying analysis. Addition of a His tag that is removable by the proteolytic activity of the polypeptides disclosed herein falls into category 1; R105H mutant appears to improve yield by ˜2-fold, placing this mutation into category 2; and mutations in positions 171-174 place these mutants into category 3.

Kuma010 with Kuma010-Cleavable His Tag MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQPGSSGSSGSQPQLPYGSSGSSGSHHHHHH (SEQ ID NO: 43 (unprocessed), SEQ ID NO: 113 (processed)) Kuma011-R105H MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 44 (unprocessed), SEQ ID NO: 114 (processed)) Kuma011-P171A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARAHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 45 (unprocessed), SEQ ID NO: 115 (processed)) Kuma011-P171R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 46 (unprocessed), SEQ ID NO: 116 (processed)) Kuma011-P171S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARSHFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFL DAAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVAS GGRIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSG RGVPDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLG KAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGS PIGVRLLQALLPSASQPQP (SEQ ID NO: 47 (unprocessed), SEQ ID NO: 117 (processed)) Kuma011-H172A MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPAFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 48 (unprocessed), SEQ ID NO: 118 (processed)) Kuma011-H172R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 49 (unprocessed), SEQ ID NO: 119 (processed)) Kuma011-H172S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPSFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 50 (unprocessed), SEQ ID NO: 120 (processed)) Kuma011-F173R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHRRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 51 (unprocessed), SEQ ID NO: 121 (processed)) Kuma011-F173S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHSRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 52 (unprocessed), SEQ ID NO: 122 (processed)) Kuma011-R174S MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARPHFSMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPEGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 53 (unprocessed), SEQ ID NO: 123 (processed)) Kuma012: P171R; H172R MSDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQ RAGELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHG LALDRANVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVP ASIAPMIEAVLGLDTRPVARRRFRMQRRAEGGFEARSQA AAPTAYTPLDVAQAYQFPEGLDGQGQCIAIIELGGGYDEASLAQYFASLG VPAPQVVSVSVDGASNQPTGDPKGPDGEVELDIEVAGALAPGAKFAVYFA PDTTAGFLDAITTAIHDPTLKPSVVSISWSGPEDSWTSAAIAAMNRAFLD AAALGVTVLAAAGDSGSTGGEQDGLYHVHFPAASPYVLACGGTRLVASGG RIAQETVWNDGPDGGATGGGVSRIFPLPAWQEHANVPPSANPGASSGRGV PDLAGNADPATGYEVVIDGEATVIGGTSAVAPLFAALVARINQKLGKAVG YLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVR LLQALLPSASQPQP (SEQ ID NO: 54 (unprocessed), SEQ ID NO: 124 (processed))

As used throughout the present application, the term “polypeptide” is used in its broadest sense to refer to a sequence of subunit amino acids, whether naturally occurring or of synthetic origin. The polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids. The polypeptides described herein may be chemically synthesized or recombinantly expressed. The polypeptides may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, or glycosylation. Such linkage can be covalent or non-covalent as is understood by those of skill in the art. The polypeptides may be linked to any other suitable linkers, including but not limited to any linkers that can be used for purification or detection (such as FLAG or His tags).

In another aspect, the present invention provides isolated nucleic acids encoding the polypeptide of any aspect or embodiment of the invention. The isolated nucleic acid sequence may comprise RNA or DNA. As used herein, “isolated nucleic acids” are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention.

In a further aspect, the present invention provides nucleic acid expression vectors comprising the isolated nucleic acid of any embodiment of the invention operatively linked to a suitable control sequence. “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the invention is intended to include other expression vectors that serve equivalent functions, such as viral vectors.

In another aspect, the present invention provides recombinant host cells comprising the nucleic acid expression vectors of the invention. The host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected or transduced. Such transfection and transduction of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.). A method of producing a polypeptide according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide. The expressed polypeptide can be recovered from the cell free extract, cell pellet, or recovered from the culture medium. Methods to purify recombinantly expressed polypeptides are well known to the man skilled in the art.

In a further aspect, the present invention provides pharmaceutical compositions, comprising the polypeptide, nucleic acid, nucleic acid expression vector, and/or the recombinant host cell of any aspect or embodiment of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can be used, for example, in the methods of the invention described below. The pharmaceutical composition may comprise in addition to the polypeptides, nucleic acids, etc. of the invention (a) a lyoprotectant, (b) a surfactant: (c) a bulking agent; (d.) atonicity adjusting agent; (e) stabilizer; (f) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, Jia-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosinotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

The polypeptides, nucleic acids, etc. of the invention may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.

The pharmaceutical compositions described herein generally comprise a combination of a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions are substantially free of non-pharmaceutically acceptable components, i.e., contain amounts of non-pharmaceutically acceptable components lower than permitted by US regulatory requirements at the time of filing this application. In some embodiments of this aspect, if the compound is dissolved or suspended in water, the composition further optionally comprises an additional pharmaceutically acceptable carrier, diluent, or excipient. In other embodiments, the pharmaceutical compositions described herein are solid pharmaceutical compositions (e.g., tablet, capsules, etc.).

The compositions described herein could also be provided as a dietary supplement as described by the US regulatory agencies.

These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by any suitable route. In a preferred embodiment, the pharmaceutical compositions and formulations are designed for oral administration. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

The pharmaceutical compositions can be in any suitable form, including but not limited to tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

In another aspect, the present invention provides methods for treating celiac sprue or non-celiac gluten sensitivity (NCGS), comprising administering to an individual with celiac sprue or NCGS an amount effective to treat the celiac sprue or NCGS of one or more polypeptides selected from the group consisting of the polypeptides of the of the invention, or using one or more of these polypeptides to process food for consumption by individuals with celiac sprue or NCGS

The inventors of the present invention have discovered that the polypeptides of the invention are capable of degrading proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ believed responsible for the bulk of the immune response in most celiac sprue patients. The polypeptides of the present invention show superior activity in degrading peptides having a PQLP (SEQ ID NO: 65) or PQQP (SEQ ID NO: 66) motif (such as PFPQPQLPY (SEQ ID NO: 67) and/or PFPQPQQPF (SEQ ID NO: 68)), which are substrates representative of gliadin) at pH 4 compared to Kuma010/011 and other polypeptides disclosed as useful for treating celiac sprue (WO2015/023728). Thus, the polypeptides of the invention constitute significantly improved therapeutics for treating celiac sprue and NCGS.

Celiac sprue (also known as celiac disease or gluten intolerance) is a highly prevalent disease in which dietary proteins found in wheat, barley, and rye products known as ‘glutens’ evoke an immune response in the small intestine of genetically predisposed individuals. The resulting inflammation can lead to the degradation of the villi of the small intestine, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue, weight loss, abdominal pain, bloating, excessive gas, indigestion, constipation, abdominal distension, nausea/vomiting, anemia, bruising easily, depression, anxiety, growth delay in children, hair loss, dermatitis, missed menstrual periods, mouth ulcers, muscle cramps, joint pain, nosebleeds, seizures, tingling or numbness in hands or feet, delayed puberty, defects in tooth enamel, and neurological symptoms such as ataxia or paresthesia. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its' prevalence in the US and Europe is estimated at 0.5-1.0% of the population. In addition to celiac sprue, a significant fraction of the population is thought to suffer from the condition of non-celiac gluten sensitivity (NCGS), which is caused by the ingestion of gluten but is mechanistically distinct from celiac disease, though the symptoms are frequently indistinguishable from those of celiac sprue.

As used herein, “treating celiac sprue or NCGS” means accomplishing one or more of the following: (a) reducing the severity of celiac sprue or NCGS; (b) limiting or preventing development of symptoms characteristic of celiac sprue or NCGS; (c) inhibiting worsening of symptoms characteristic of celiac sprue or NCGS; (d) limiting or preventing recurrence of celiac sprue or NCGS in patients that have previously had the disorder; (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for celiac sprue or NCGS; and (f) limiting development of celiac sprue or NCGS in a subject at risk of developing celiac sprue or NCGS, or not yet showing the clinical effects of celiac sprue or NCGS.

The individual to be treated according to the methods of the invention may be any individual suffering from celiac sprue or NCGS, including human subjects. The individual may be one already suffering from symptoms or one who is asymptomatic.

As used herein, an “amount effective” refers to an amount of the polypeptide that is effective for treating celiac sprue. The polypeptides are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including orally, parentally, by inhalation spray, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. In a preferred embodiment, the pharmaceutical compositions and formulations are orally administered, such as by tablets, pills, lozenges, elixirs, suspensions, emulsions, solutions, or syrups.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). A suitable dosage range may, for instance, be 0.1 ug/kg-100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight. The polypeptides can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.

EXAMPLE 1

Gliadin is highly enriched in proline (P) and glutamine (Q), which renders it recalcitrant to degradation by human digestive enzymes. PQ-rich peptide fragments derived from partial digestion of gliadin are deamidated in the intestinal lumen, thereby allowing binding to HLA-DQ2 or DQ8, and stimulation of a Th1 inflammatory response in people with CD³. The gliadin endopeptidase KUMAMAX™ (hereafter referred to as Kuma011, or Kuma 010 if referring to Kuma011 including the C-terminal histidine tag: GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139)), which demonstrates stability and functionality in gastric conditions, was previously engineered to break down peptides containing the PQ dipeptide motif. Based on the crystal structure of Kuma010 (PDB ID 4NE7), we redesigned the active site of Kuma010 selecting for mutations to increase activity against immunogenic gliadin peptides. Designed mutants were then screened for increased activity against the highly immunogenic 33mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69)) and 26mer (FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70)) gliadin peptides^(6,7). These peptides harbor either the PQL or PQQ tripeptide motif, representative of all gliadin T cell epitopes shown to be toxic for the vast majority of celiac patients⁸. In this manner, the variant Kuma030 was built. Kuma030 is 44-fold more active against peptides containing PQQ, and 11-fold more active against peptides containing PQL, than Kuma010.

Based on the molecular modeling, the putative S1′ peptide binding interface of Kuma010 consists of entirely hydrophobic residues, and should therefore prefer hydrophobic residues such as leucine, and not polar residues such as glutamine, at P1′. The S1′ binding pocket of Kuma030 introduces an isoleucine to threonine mutation (I463T), which is predicted to provide a hydrogen bond with a P1′ glutamine, enabling this enzyme to accommodate both leucine and glutamine in the S1′ subsite and thereby target both PQL and PQQ tripeptides. Kuma030 also incorporates six additional mutations (K262E, E269T, S354Q, G358S, D399Q, A449Q) that provide enhanced catalytic efficiency on the 26mer and 33mer peptides. G358S is predicted to stabilize the loop containing an engineered histidine introduced in Kuma010 which is predicted to hydrogen bond to the P1 glutamine residue. The remaining mutations are predicted to stabilize the protein structure as modeled.

While dozens of PQ-rich epitopes have been linked to CD, several peptides derived from gliadin (wheat), hordein (barley), and secalin (rye) have been shown to account for the vast majority of the immune response in CD and have thus been classified as immunodominant⁸. In wheat, these include the peptides W02-E07 (LQPFPQPQLPYPQPQ (SEQ ID NO: 133)), W03-E07 (QPFPQPQQPFPWQP (SEQ ID NO: 134)), and the 33mer peptide, which contains the W02-E07 sequence^(6,9). These peptides harbor several epitopes shown to be highly immunogenic⁹⁻¹¹. To evaluate the ability of Kuma030 to destroy these epitopes throughout gluten, purified whole gluten was incubated with Kuma030 under simulated gastric conditions (pH 4.0 at 37° C. with 0.6 mg mL⁻¹ pepsin)¹². The gluten fraction remaining after degradation was quantified using ELISA assays based on either the R5 or G12 antibodies recognizing the amino acid motifs QQPFP (SEQ ID NO: 135) and QPQLPY (SEQ ID NO: 136), respectively, which encompass all of the immunodominant epitopes in the above peptides^(13,14). To compare the activity of Kuma030 to that of published glutenases, we also examined the glutenases EPB2 and SCPEP, which are currently being pioneered at a 1:1 ratio as a combination enzyme therapeutic for CD¹⁵. The EPB2 and SCPEP enzymes generated in this work were verified to have activities consistent with that of published values^(16,17). Upon incubation with gluten, we observed a dose-dependent reduction in the QQPFP (SEQ ID NO: 135) or QPQLPY (SEQ ID NO: 136) load using either Kuma030, Kuma010, or a 1:1 combination of EPB2 and SCPEP (FIG. 1A). At a 1:25 w:w ratio of enzyme:gluten, EPB2 and SCPEP broke down 84.8% of the gluten present, consistent with previously published reports of this enzyme combination^(18,19). Kuma030 was highly effective at eliminating these peptide epitopes throughout gluten, with a 1:400 w:w ratio of enzyme:gluten being sufficient to reduce the immunogenic gluten present by over 99.5% at 60 minutes, as quantified by both methods used (FIG. 1A). Kuma030-dependent reduction in immunogenic gluten load is rapid, with >98% degradation achieved by 5 minutes at a w:w ratio of 1:25 (FIG. 1B). Mass spectroscopy of the Kuma030 cleavage products revealed that Kuma030 cleaved each peptide after the PQ dipeptide motif in the immunodominant epitopes from wheat (33mer, W02-E07, and W03-E07), and also those from barley B08-E2E7 (PQQPIPQQPQPYPQQ (SEQ ID NO: 61)) and rye R11-E2E7 (QPFPQQPEQIIPQQP)⁹ (SEQ ID NO: 62) (FIG. 1C). While these intact peptides are highly immunostimulatory, the peptide breakdown products are not predicted to stimulate the immune system, since Kuma030 action results in elimination of the core 9mer epitope thought to trigger the immune response²⁰. The ability of Kuma030 to cleave these peptides containing either a PQL or a PQQ tripeptide motif, and to reduce the gliadin load as measured by both G12 and R5 antibodies, are consistent with the hypothesis that Kuma030 can bind and cleave peptides with either a leucine or a glutamine in the S1′ binding pocket.

The ability of Kuma030 to efficiently degrade immunogenic gliadin epitopes suggests that incubation of gliadin with Kuma030 might reduce its capacity to stimulate a T-cell mediated immune response. T cell assays utilizing cells derived from the intestinal biopsies of celiac patients represent the gold standard for this evaluation. To directly evaluate the hypothesis that incubation with Kuma030 would decrease or eliminate the immunostimulatory capacity of gliadin, we performed T cell assays in which cells were exposed to Kuma030-treated gliadin and the resulting T cell reaction was assessed. The highly gliadin-reactive intestinal CD4⁺ T cell lines used in this study were previously generated from intestinal mucosa and have been shown to react to a diversity of epitopes across different gliadin families²¹. Kuma030 and pepsin were incubated with purified wheat gliadin in laboratory-simulated gastric conditions for 60 minutes. In order to mimic transit into the intestinal compartment, the pH levels of the samples were then increased, and the samples were treated with chymotrypsin and deamidated with TG2 to unmask the immunogenic epitopes. The resulting gliadin samples were presented to T cell lines, and stimulation was assessed by measuring IFN-γ production (FIGS. 2A-E) and T cell proliferation (FIG. 3A-D). Exposure of T cell lines to pepsin-treated gliadin resulted in stimulation of production of IFN-γ, and co-treatment of gliadin with pepsin and Kuma030 reduced this response, eliminating it at higher concentrations. The decreases observed in IFN-γ production were not due to Kuma030 toxicity. Importantly, Kuma030 eliminated the T cell response to gliadin in every T cell line tested regardless of T cell epitope specificity, indicating that Kuma030 is effective against all epitopes recognized by the T cell lines used in this assay. Since these epitopes span the three major gliadin families: α-, ω-, and γ-gliadin, this suggests that Kuma030 is capable of degrading immunogenic epitopes within all relevant regions of gliadin.

The experiments above demonstrate the ability of Kuma030 to degrade immunogenic gliadin epitopes in the context of purified whole gluten or gliadin. However, to assess practical application, it is important to evaluate Kuma030 effectiveness in physiologically relevant food and beverage matrices. To assess the activity of Kuma030 in gastric digestion scenarios, we tested the ability of Kuma030 to break down gluten in an acidified bread slurry and in a wheat beer. Whole-wheat bread was mashed in artificial saliva to simulate mastication at a bread concentration representative of that in the stomach after ingestion of one slice of bread. The mixture was then acidified by the addition of HCl and pepsin, and glutenases at various concentrations were added. The amount of gluten remaining was then quantified after 30 minutes of digestion, which represents the average lag time of food in the stomach before the commencement of ingesta release into the duodenum through the pyloric opening²². At the highest concentration of glutenase tested (1000 μg mL⁻¹), treatment with EPB2 and SCPEP resulted in84.4% gluten degradation (FIG. 4A). This is comparable to published results from a Phase I study showing that EPB2 and SCPEP eliminate 70-79% of gluten in a test meal at a 1:10 enzyme:gluten ratio in the human stomach after a 30 minute incubation time²³. At a concentration of 62.5 μg mL⁻¹ (a 1:160 w:w ratio), Kuma030 reduced the level of gluten in the bread to below 20 ppm (the FDA threshold for “gluten free” labeling). Finally, the gluten-degrading ability of Kuma030 was tested directly in a wheat beer, since wheat beers demonstrate comparatively high gluten levels²⁴. Beer was incubated with Kuma030 at 37° C. or 4° C. at two enzyme concentrations. Samples were taken at various timepoints and the concentration of remaining gluten was quantified. We found that incubation of beer, which demonstrated a gluten concentration of ˜764 ppm, with Kuma030 decreased the gluten level to below 20 ppm by only 5 minutes (FIG. 4B). The rapidity of this effect is particularly important since liquids empty through the stomach considerably faster than solids²⁵. Surprisingly, Kuma030 significantly reduced the gluten load of wheat beer even at 4° C., as incubation with the Kuma030 at 700 μg mL⁻¹ reduced the gluten levels to below 20 ppm by 5 minutes.

At this time, the only therapy for celiac disease is a lifelong strict gluten free diet. Oral enzyme therapy has been considered an attractive treatment option for CD since the identification of PQ-rich immunogenic gliadin epitopes that stimulate the immune response³. A useful characteristic of any oral enzyme therapeutic for CD is the ability to break down immunogenic peptides in gastric conditions, since the inflammatory immune response to gliadin occurs immediately upon entering the intestine²⁶. Gluten challenge studies in CD patients have shown that the ingested gluten load must be kept at 10 mg or less in order to prevent intestinal damage^(27,28). Indeed, the FDA currently mandates that any food labeled as “gluten free” must demonstrate less than 20 ppm gluten, since strict adherence to this standard is predicted to result in a daily ingestion of 10 mg or less. Thus, the accidental ingestion of 1 g of gluten (approximately the amount of gluten present in a crouton) must be reduced by 99% or greater in the gastric compartment in order to prevent the intestinal damage and symptoms that would arise from gluten exposure. There is therefore a clear need for glutenases that can rapidly destroy immunogenic gliadin epitopes in gastric conditions. In a bread slurry model representing ingestion of 4 g of gluten, Kuma030 was found to degrade >99.8% of the gliadin load in 30 minutes at a 1:160 w:w ratio. Additionally Kuma030 specifically destroys peptides with the PQ dipeptide motif, which is commonly found throughout the immunogenic regions of gluten. Indeed, Kuma030 is capable of degrading all immunodominant peptides tested, and gliadin treated with Kuma030 failed to stimulate IFN-γ production by all T cell lines tested, which is significant since CD patients demonstrate a myriad of responses to different immunogenic epitopes.

REFERENCES FOR EXAMPLE 1

-   1. Rubio-Tapia, A., Ludvigsson, J. F., Brantner, T. L.,     Murray, J. A. & Everhart, J. E. The prevalence of celiac disease in     the United States. Am J Gastroenterol 107, 1538-1544; quiz 1537,     1545 (2012). -   2. Catassi, C., Gatti, S. & Lionetti, E. World perspective and     celiac disease epidemiology. Dig Dis 33, 141-146 (2015). -   3. Sollid, L. M. Coeliac disease: dissecting a complex inflammatory     disorder. Nat Rev Immunol 2, 647-655 (2002). -   4. Gordon, S. R., et al. COMPUTATIONAL DESIGN OF AN alpha-GLIADIN     PEPTIDASE. J Am Chem Soc (2012). -   5. Richter, F., Leaver-Fay, A., Khare, S. D., Bjelic, S. & Baker, D.     De novo enzyme design using Rosetta3. PLoS One 6, e19230 (2011). -   6. Shan, L., et al. Structural basis for gluten intolerance in     celiac sprue. Science 297, 2275-2279 (2002). -   7. Shan, L. Identification and analysis of multivalent     proteolytically resistant peptides from gluten: implications for     celiac sprue. Journal of Proteome Research (2005). -   8. Sollid, L. M., Qiao, S. W., Anderson, R. P., Gianfrani, C. &     Koning, F. Nomenclature and listing of celiac disease relevant     gluten T-cell epitopes restricted by HLA-DQ molecules.     Immunogenetics 64, 455-460 (2012). -   9. Tye-Din, J. A., et al. Comprehensive, quantitative mapping of T     cell epitopes in gluten in celiac disease. Sci Transl Med 2, 41ra51     (2010). -   10. Arentz-Hansen, H., et al. The intestinal T cell response to     alpha-gliadin in adult celiac disease is focused on a single     deamidated glutamine targeted by tissue transglutaminase. J Exp Med     191, 603-612 (2000). -   11. Arentz-Hansen, H., et al. Celiac lesion T cells recognize     epitopes that cluster in regions of gliadins rich in proline     residues. Gastroenterology 123, 803-809 (2002). -   12. Chang, J. H., et al. A novel placement method of the Bravo     wireless pH monitoring capsule for measuring intragastric pH. Dig     Dis Sci 54, 578-585 (2009). -   13. Lupo, A., Roebuck, C., Walsh, A., Mozola, M. & Abouzied, M.     Validation study of the Veratox R5 rapid ELISA for detection of     gliadin. J AOAC Int 96, 121-132 (2013). -   14. Moron, B., et al. Sensitive detection of cereal fractions that     are toxic to celiac disease patients by using monoclonal antibodies     to a main immunogenic wheat peptide. Am J Clin Nutr 87, 405-414     (2008). -   15. Landeaho, M. L., et al. The Glutenase ALV003 Attenuates     Gluten-Induced Mucosal Injury in Patients with Celiac Disease.     Gastroenterology (2014). -   16. Bethune, M. T., Strop, P., Tang, Y., Sollid, L. M. & Khosla, C.     Heterologous expression, purification, refolding, and     structural-functional characterization of EP-B2, a self-activating     barley cysteine endoprotease. Chem Biol 13, 637-647 (2006). -   17. Ehren, J., Govindarajan, S., Moron, B., Minshull, J. &     Khosla, C. Protein engineering of improved prolyl endopeptidases for     celiac sprue therapy. Protein Eng Des Sel 21, 699-707 (2008). -   18. Siegel, M., et al. Rational design of combination enzyme therapy     for celiac sprue. Chem Biol 13, 649-658 (2006). -   19. Gass, J., Bethune, M. T., Siegel, M., Spencer, A. & Khosla, C.     Combination enzyme therapy for gastric digestion of dietary gluten     in patients with celiac sprue. Gastroenterology 133, 472-480 (2007). -   20. Petersen, J., et al. T-cell receptor recognition of     HLA-DQ2-gliadin complexes associated with celiac disease. Nat Struct     Mol Biol 21, 480-488 (2014). -   21. Camarca, A., et al. Intestinal T cell responses to gluten     peptides are largely heterogeneous: implications for a peptide-based     therapy in celiac disease. J Immunol 182, 4158-4166 (2009). -   22. Pera, P., et al. Influence of mastication on gastric emptying. J     Dent Res 81, 179-181 (2002). -   23. Siegel, M., et al. Safety, tolerability, and activity of ALV003:     results from two phase 1 single, escalating-dose clinical trials.     Dig Dis Sci 57, 440-450 (2012). -   24. Picariello, G., et al. Proteomics, peptidomics, and immunogenic     potential of wheat beer (weissbier). J Agric Food Chem 63, 3579-3586     (2015). -   25. Houghton, L. A., et al. Relationship of the motor activity of     the antrum, pylorus, and duodenum to gastric emptying of a     solid-liquid mixed meal. Gastroenterology 94, 1285-1291 (1988). -   26. Castillo, N. E., Theethira, T. G. & Leffler, D. A. The present     and the future in the diagnosis and management of celiac disease.     Gastroenterol Rep (Oxf) 3, 3-11 (2015). -   27. Catassi, C., et al. A prospective, double-blind,     placebo-controlled trial to establish a safe gluten threshold for     patients with celiac disease. Am J Clin Nutr 85, 160-166 (2007). -   28. Akobeng, A. K. & Thomas, A. G. Systematic review: tolerable     amount of gluten for people with coeliac disease. Aliment Pharmacol     Ther 27, 1044-1052 (2008).

EXAMPLE 2

Computationally designed enzymes were then produced and tested for their ability to break down immunogenic gliadin peptides. Mutations that were shown to improve the ability of the enzyme to target relevant peptides were then combined and tested in an iterative process to further increase activity. More recently, design has been extended to the S1′ binding pocket to prefer either L or Q amino acids. This engineering effort has greatly increased activity on peptides containing a PQL or PQQ tripeptide, which are located within the core epitope of virtually all immunogenic gliadin peptides.

Several Kuma010 variants are used. The specific mutational differences, and their relative effects on activity, are listed in Table 6 below.

TABLE 6 Activity Improvement Variant Background^(a) Mutation^(b) over Background^(c) Kuma010 Kumamolisin-As V119D 116x S262K N291D D293T G319S D358G D368H Kuma020 Kuma010 G358S 7-19x (PQL) I463T 15-35x (PQQ) Kuma021 Kuma020 D399Q 1-2x (PQL) A449Q 1.2-2.5x (PQQ) Kuma030 Kuma021 K262E 1.2-2x (PQL) E267T 1.2-2x (PQQ) S354Q Kuma031 Kuma030 GSTENLYFQSGALEHHHHHH No change (PQL); (SEQ ID NO: 139) production mutant Deleted from C-terminus Kuma032 Kuma031 P171R No change (PQL)); H172R production mutant Kuma040 Kuma010 S319A 35-50x (PQL) H368F 30-40x (PQQ); D399Q Kuma030 is 2-fold A449Q more active on I463T PQQ, and 2-fold less active on PQL, than Kuma040 Kuma041 Kuma040 GSTENLYFQSGALEHHHHHH 1.0-1.2x (PQL) (SEQ ID NO: 139) Deleted from C-terminus Kuma042 Kuma041 P171R 1.2-1.5x (PQL) H172R Kuma050 Kuma021 K262E 1.5-2x (PQL) E267T 0.01-0.2x (PQQ) S354Q L270V T463I Kuma060 Kuma030 G320M In bread assays. Kuma060 is about twice as active as Kuma030 Kuma061 Kuma060 GSTENLYFQSGALEHHHHHH about same (SEQ ID NO: 139) activity as Deleted from C-terminus Kuma060. Kuma062 Kuma061 P171R ND^(d); about same H172R activity as Kuma060. Kuma070 Kuma040 G320M 2-3x (PQL) 2-4x (PQQ) Kuma071 Kuma070 GSTENLYFQSGALEHHHHHH ND^(d;) about 0.2X as (SEQ ID NO: 139) active as Kuma Deleted from C-terminus 070 Kuma072 Kuma070 GSTENLYFQSGALEHHHHHH about 0.7X as (SEQ ID NO: 139) active as Kuma Deleted from C-terminus 070; P171R H172R ^(a)The specific Kuma010 variant that served as a background upon which the listed mutations were made. ^(b)Mutational positions are denoted relative to the full-length Kuma010 enzyme. ^(c)Activity Improvement was calculated for each variant as a fold increase in activity relative to that of the “background” enzyme, the template enzyme used to make that variant. Activity was measured on one or more of the following substrates: a fluorescent-labeled PQPQLP (SEQ ID NO: 156) substrate, the 33mer⁵ (contains PQL) or 26mer⁶ (contains PQQ) peptides, or DQ2.5-glia-α1a (contains PQL) or DQ2.5-glia-ω1 (contains PQQ)⁷. It is important to note that since hundreds of Kuma010 variants were testes, it would have been impractical to obtain kinetic constants for each mutant, thus the fold improvement numbers shown here are estimates instead of definitive numbers. The fold improvement numbers presented here are calculated from the amount of peptide degradation product detected in the degradation assay by LC-MS. ^(d)ND: not determined. The activities of leads Kuma060, Kuma061, and Kuma062; and Kuma070, Kuma071, and Kuma072; were compared directly to each other in bread or meal degradation assays instead of by assessment of individual peptide degradation in order to confirm that no decrease in enzymatic activity occurred upon loss of the His tag. The ability of Kuma070 to break down PQL- and PQQ-containing peptides was compared to Kuma040 as discussed below. Kuma020, Kuma021, and Kuma030

The active site of Kuma010 underwent further design effort to improve activity against substrates containing either PQL or PQQ tripeptides. Engineering efforts identified the mutations G358S and I463T as important contributors to increased activity. The G358S mutation was a refinement to a previous mutation made at this site in Kuma010. The I463T mutation eliminated the steric hindrance encountered in the P1′ binding pocket when targeting PQL tripeptide motifs, and introduced a new predicted hydrogen bond when PQQ was the substrate⁴. The Kuma010 variant harboring these two mutations demonstrated a very large improvement over the original Kuma010 and was named Kuma020.

Additional mutations were made to this Kuma020 enzyme. D399Q and A449Q were mutations that were located outside of the active site, and so are not predicted to affect binding to the substrate. Instead, these two mutations resulted in new predicted intramolecular hydrogen bonds and were thus predicted to stabilize the enzyme. The resulting variant, Kuma021, showed a further increase in activity.

Three other refinements contributed to the generation of Kuma030 from Kuma021. Kuma030 is described in detail above.

Kuma040 and Kuma050

As an alternative to the mutations listed above, a different set of mutations on the Kuma010 background, S319A and H368F, led to a different active site architecture than the one found in Kuma030. Together these mutations, along with D399Q, A449Q, and I463T, (mutations that also increased activity in Kuma030) make up the Kuma040 variant. Kuma041, Kuma042, Kuma070, Kuma071, and Kuma072 have Kuma040-like active sites, while Kuma031, Kuma032, Kuma060, Kuma061, and Kuma062 have Kuma030-like active sites.

Kuma050 is a Kuma010 variant built on the Kuma021 background with an active site architecture that has more in common with Kuma030 than Kuma040. However, Kuma050 lacks the I463T mutation, and instead harbors a L270V mutation which is predicted to increase its activity against PQL-containing peptides but is predicted to hinder glutamine from accessing the P1′ binding pocket, thereby decreasing activity on PQQ-containing substrates. Accordingly, Kuma050 specifically demonstrates a high level of activity against substrates containing PQL, but not against substrates containing PQQ. The specificity profile of Kuma050 was desired due to the fact that several studies have indicated that the immunodominant 33mer peptide from α-gliadin, which contains several PQL motifs and no PQQ motifs, may be the peptide responsible for the vast majority of the disease in a subset of patients.

The activities of Kuma010, Kuma020, Kuma030, Kuma040, and Kuma050 are shown below for the highly immunogenic 33mer peptide of α-gliadin (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 69)) and the 26mer peptide of γ-gliadin (FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 70)), which represent PQL- and PQQ-containing peptides, respectively. The appearance of breakdown products over time (LPYPQPQPF (SEQ ID NO: 137) for 33mer peptide; FLQPQ (SEQ ID NO: 138) for 26mer peptide) are shown in FIGS. 5A-B.

In both cases, Kuma030 was the dominant enzyme tested, though Kuma040 demonstrated activity almost as potent as Kuma030 especially against the 26mer peptide. As expected, Kuma050 demonstrated good activity on the 33mer peptide, but very poor activity on the 26mer peptide. Compared to Kuma030 and Kuma040, Kuma020 demonstrated a moderate amount of activity on both peptides, consistent with it being a design intermediate as shown above. All variants demonstrate a marked improvement on the original Kuma010 enzyme.

Kuma060 and Kuma070

An additional designed mutation was the mutation G320M. This mutation did not appear particularly promising as it was not predicted to greatly improve activity, and indeed, this residue, while in the active site, does not appear to make direct contact with the gliadin substrate. However, mutation at this position improved activity by 2-4 fold on both substrates. This could be due to slight changes in the Kuma010 backbone incurred by incorporation of the methionine, rendering it into a confirmation that is even more favorable for catalysis. The G320M mutation was incorporated into the Kuma030 and Kuma040 backgrounds to generate enzymes Kuma060 and Kuma070, respectively.

FIGS. 6A-B show the improvement in activity attributed to the G320M mutation on the Kuma040 background in degradation of both immunogenic epitopes DQ2.5-glia-α1a (contains PQL), and DQ2.5-glia-ω1 (contains PQQ).

The KumaXX1 and KumaXX2 Series

With the exception of Kuma021, Kuma010 variants labeled KumaXX1 (ex. Kuma031) correspond to the KumaXX0 variant (ex. Kuma030) in which the C-terminal TEV protease cut site and 6×His tag have been genetically deleted (GSTENLYFQSGALEHHHHHH (SEQ ID NO: 139)). This tag, which was originally added to the Kumamolisin-As enzyme for high-throughput, easy purification of Kuma010 variants, was removed in certain Kuma010 lead variants as the 6× His tag is not preferable on biologic pharmaceuticals. In general, the removal of this tag did not affect the activity of the enzyme, though His tag removal did appear to result in a slight decrease in the ability of the Kuma070 enzyme (but not the Kuma060 enzyme) to degrade gliadin in a gastric digestion of whole wheat bread.

Kuma010 variants labeled KumaXX2 (ex Kuma032) also lack the His tag, and contain the following additional mutations: P171R and H172R. These mutations don't affect activity of the enzyme, but were incorporated to simplify the purification process. These mutations were introduced into the propeptide domain in the N-terminal region of Kuma010. Since these mutations lie within the propeptide domain, they are not present in the mature, active enzyme. These two mutations fall within the N-terminal region of the protein that sits in the enzyme's active site before cleavage upon exposure to low pH. Since this region is in proximity of the catalytic residues, it is hypothesized that this is the region that undergoes the initial cleavage event upon purification of the Kuma010 enzyme during standard purification procedures. The partially cleaved enzyme N-terminus remains tightly associated with the mature enzyme until the enzyme is exposed to acid. While this initial self-processing during protein purification does not negatively affect activity, it can complicate interpretation of SDS-PAGE analysis by individuals unfamiliar with the Kuma010 enzyme. Thus, in order to simplify the SDS-PAGE profile of purified enzyme, the P171R and H172R mutations were incorporated to reduce the amount of initial N-terminal cleavage that occurs during the protein purification process.

Since KumaXX1 and KumaXX2 variants lack a His tag, they are not purified by Ni affinity chromatography. Instead, these variants are purified by anion exchange chromatography. The following graph demonstrates activity of variants Kuma030, Kuma031, Kuma032, Kuma040, Kuma041, and Kuma042 on immunogenic gliadin peptide DQ2.5-glia-α1a. In this case, all proteins were purified by anion exchange chromatography (even Kuma030 and Kuma040 which harbor an intact 6× Histidine tag), for the sake of comparison. As shown in FIG. 7, P171R and H172R mutations do not appear to negatively affect activity as Kuma032 and Kuma042 demonstrate comparable activity as Kuma030 and Kuma040 (Kuma042 even shows a slight increase in activity against this substrate compared to Kuma040).

Kuma010 variant Kuma062 demonstrates a high amount of activity and it lacks a His tag. A comparison of Kuma062 with Kuma030 and Kuma040 is shown in FIGS. 8A-B (left, detection of full-length DQ2.5-glia-ω1; right, detection of breakdown product):

Enzyme Kinetics

Biochemical parameters were estimated for: Kuma010, Kuma030, Kuma040, and Kuma050. These were estimated using immunogenic gliadin epitopes DQ2.5-glia-α1a and DQ2.5-glia-ω1. Degradation assays were performed with 100 nM enzyme at 37° C. in 100 mM NaOAc pH 4.0 buffer. The below table shows the initial velocity of the degradation reaction as a function of substrate concentration. k_(cat) and K_(M) were calculated from this using the Michaelis-Menten equation.

The biochemical parameters of all tested enzymes are shown in Table 7 below.

TABLE 7 Enzyme [Enzyme] Substrate Vmax (M s⁻¹) Km (mM) Kcat (s⁻¹) kcat/Km (M⁻¹ s⁻¹) Kuma010 100 nM Glia_α1 3.70E−07  4 6 mM 3.7 819 Kuma030 100 nM Glia_α1 1.74E−06  1.9 mM 17.4 9034 Kuma040 100 nM Glia_α1 1.49E−06 0.86 mM 16.3 19109 Kuma050 100 nM Glia_α1 1.42E−06  2.5 mM 14.2 5613 Kuma010 100 nM Glia_α1 1.22E−07 16.4 mM 1.2 74 Kuma030 100 nM Glia_α1 2.73E−06  8.4 mM 27.3 3268 Kuma040 100 nM Glia_α1 2.46E−06 15.3 mM 24.6 1603 Kuma050 100 nM Glia_α1 2.78E−06  5.0 mM 27.8 56

The mutations made to Kuma030 and Kuma040 greatly increase activity against these peptides, which is promising for their use in detoxifying peptides that are linked to celiac disease. As predicted, the mutations made to Kuma050 increased activity on the DQ2.5-glia-α1a peptide, but not on the DQ2.5-glia-ω1 peptide.

REFERENCES FOR EXAMPLE 2

(1) Gordon, S. R.; Stanley, E. J.; Wolf, S.; Toland, A.; Wu, S. J.; Hadidi, D.; Mills, J. H.; Baker, D.; Pultz, I. S.; Siegel, J. B. Journal of the American Chemical Society 2012.

(2) Wlodawer, A.; Li, M.; Gustchina, A.; Tsuruoka, N.; Ashida, M.; Minakata, H.; Oyama, H.; Oda, K.; Nishino, T.; Nakayama, T. J Biol Chem 2004, 279, 21500.

(3) Leaver-Fay, A.; Tyka, M.; Lewis, S. M.; Lange, O. F.; Thompson, J.; Jacak, R.; Kaufman, K.; Renfrew, P. D.; Smith, C. A.; Sheffler, W.; Davis, I. W.; Cooper, S.; Treuille, A.; Mandell, D. J.; Richter, F.; Ban, Y. E.; Fleishman, S. J.; Corn, J. E.; Kim, D. E.; Lyskov, S.; Berrondo, M.; Mentzer, S.; Popovic, Z.; Havranek, J. J.; Karanicolas, J.; Das, R.; Meiler, J.; Kortemme, T.; Gray, J. J.; Kuhlman, B.; Baker, D.; Bradley, P. Methods Enzymol 2011, 487, 545.

(4) Wolf, C.; Siegel, J. B.; Tinberg, C.; Camarca, A.; Gianfrani, C.; Paski, S.; Guan, R.; Montelione, G.; Baker, D.; Pultz, I. S. Journal of the American Chemical Society 2015, 137, 13106.

(5) Shan, L.; Molberg, O.; Parrot, I.; Hausch, F.; Filiz, F.; Gray, G. M.; Sollid, L. M.; Khosla, C. Science 2002, 297, 2275.

(6) Shan, L. Journal of Proteome Research 2005.

(7) Sollid, L. M.; Qiao, S. W.; Anderson, R. P.; Gianfrani, C.; Koning, F. Immunogenetics 2012, 64, 455.

(8) Arentz-Hansen, H.; Korner, R.; Molberg, O.; Quarsten, H.; Vader, W.; Kooy, Y. M.; Lundin, K. E.; Koning, F.; Roepstorff, P.; Sollid, L. M.; McAdam, S. N. J Exp Med 2000, 191, 603. 

We claim:
 1. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:129.
 2. A pharmaceutical composition, comprising the polypeptide of claim 1, and a pharmaceutically acceptable carrier. 